Peripheral T cell tolerance involves deletion and hyporesponsiveness, and is due in large part to signal 1 (TCR) in the absence of signal 2 (costimulation). Although CD28 has been proposed to constitute signal 2, there is increasing evidence that other cell surface receptors are involved. We have studied one member of the TNFR family, named OX40 (CD134), whose expression is inducible on T cells. Our data show that 0X40 determines the magnitude of both primary and secondary CD4 responses. Specifically, 0X40 costimulation prolongs clonal expansion during the peak of the primary T cell response, enhances differentiation and cytokine secretion, suppresses cell death, and promotes T cell memory. Moreover, we now have new data which show that 0X40 stimulation can not only prevent the induction of CD4 tolerance, but can reverse pre-existing tolerance, targeting hyporesponsive T cells, inducing them to expand, and allowing them to regain their responsiveness. In this application we propose to delineate the mode of action of 0X40 in preventing T cell tolerance and to explore the utility of targeting 0X40 during tolerizing responses. Using in vitro and in vivo systems with TCR transgenic T cells and cloned T cells, we will determine whether 0X40 signals rescue the defects in JNK and NF-kB activation that are seen in tolerant CD4 cells and that these pathways account for reversal of hyporesponsiveness. Additionally, we will show if 0X40 action in enhancing accumulation of non-tolerant T cells is through regulating the anti-apoptotic molecules bcl-2, bcl-xL, TAP, and FLIP, and/or controlling the degradation of the cell cycle inhibitor p27kip1. We will determine whether 0X40 signals regulate tolerance of CD8 cells using adoptive transfer of TCR transgenic T cells in situations which are helper dependent or independent, and if 0X40 signals can reverse CD8 tolerance. We will investigate the role of 0X40 in induction and persistence of unresponsiveness of both CD4 and CD8 T cells using 0X40 knockout mice, and show the therapeutic potential of targeting 0X40 during tolerizing responses induced by several tumors. Lastly, we will determine the role of 0X40 in CD4 and CD8 tolerance to islet-expressed antigen in a model of autoimmune diabetes using RIP-mOVA mice. These studies will show the role of 0X40 in determining peripheral tolerance in both CD4 and CD8 compartments, and whether 0X40 is a viable target for reversing the tolerance process in disease.